title: Temperature and pressure dependence of azurin stability as monitored by tryptophan fluorescence and phosphorescence. The case of F29A mutant.
abstract: The effects of a single-point, F29A, cavity-forming mutation on the unfolding thermodynamic parameters of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift in the fluorescence spectrum, led to a volume change of 70-90mlmol(-1). The difference in the unfolding volume between F29A and wild type azurin was smaller than the volume of the space theoretically created in the mutant, indicating that the cavity is, at least partially, filled with water molecules. The complex temperature dependence of the unfolding volume, for temperatures up to 20°C, suggests the formation of an expanded form of the protein and highlights how the packing efficiency of azurin appears to contribute to the magnitude of internal void volume at any given temperature. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime. At 40°C the application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between wild type and the cavity mutant is the inversion point which happens at 300MPa for wild type and at 150MPa for F29A. This suggests that, for the cavity mutant, pressure-induced internal hydration is more dominant than any compaction of the globular fold at relatively low pressures.
authors.affiliation: Istituto di Biofisica, CNR, via G. Moruzzi 1, 56124 Pisa, Italy. Electronic address: danika.tognotti@pi.ibf.cnr.it.
authors.forename: Danika
authors.initials: D
authors.lastname: Tognotti
authors.forename: Edi
authors.initials: E
authors.lastname: Gabellieri
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Morelli
authors.forename: Patrizia
authors.initials: P
authors.lastname: Cioni
mesh_terms: D019943:Amino Acid Substitution; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011312:Pressure; D055550:Protein Stability; D058767:Protein Unfolding; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001400:Azurin; D014364:Tryptophan
keywords: Azurin; High pressure; Protein dynamics; Protein unfolding; Tryptophan fluorescence; Tryptophan phosphorescence